The proposed work will employ the recently developed, reversible thrombin probe, dansylarginine N(3-ethyl-1,5-pentanediyl)amide (DAPA) to study the influence of heparin on the inactivation of thrombin (IIa) by antithrombin-III (AT-III). DAPA is ideally suited for the proposed studies since when it binds IIa it both inhibits the enzyme and exhibits enhanced fluorescence intensity. Through its inhibitory properties it effectively "buffers" levels of active IIa and thereby reduces in a predictable fashion the rate at which AT-III inhibits the enzyme. Thus the rate-enhancing effects of heparin can be readily studied without the need for rapid-mix, stopped-flow techniques. In addition the spectral properties of the DAPA-IIa complex provide a sensitive and continuous monitor of the progress of the reaction. Electrophoretically homogeneous proteins will be used. Commercially available heparin will be fractionated first by affinity chromatography on immobilized AT-III, IIa, and platelet factor 4 to obtain functional homogeneity and, further, by gel filtration to obtain molecular weight homogeneity. The kinetics of the inactivation of IIa by At-III as measured by DAPA in the presence of various fractions of heparin will be analyzed to determine: 1) the maximum enhancement of rate obtained; and 2) apparent stoichiometries and dissociation constants chracteristic of the interactions of the various subfractions of heparin with AT-III and IIa. Heparan-sulfate-like anticoagulants previously isolated from the plasma of two patients with multiple myeloma will be studied similarly. These anticoagulants were isolated by affinity chromatography on PF-4 Sepharose. These studies are aimed at obtaining a better understanding of the anticoagulant properties of heparin and heparin-like substances, and insights into the role of inhibitors found in plasma, especially AT-III, in the regulation of blood coagulation.